DNA Transactions in Mammalian Chromosomes: Choosing the Right Partner and Staying Faithful

1. DNA Transactions in Mammalian Chromosomes: Choosing the Right Partner and Staying Faithful Alan S. Waldman Department of Biological Sciences University of South Carolina

2. Homologous Recombination: The exchange of information (nucleotides) between similar DNA sequences.

3. Recombination is involved in: • Genome maintenance -- DNA repair, replication rescue • Development of disease (cancer, age-related disorders) • Generation of genetic diversity in meiosis • Gene targeting

5. One model for homologous recombination conversion tract

6. Problem: How does a cell balance the benefits of efficient DNA recombination against the risk of deleterious rearrangements? Basic Issue: How similar must two sequences be in order to undergo recombination in mammalian chromosomes?

7. Recombination Rate ~ 10-6 homologous: tk tk homeologous: < 10-9 tk tk

8. What we have learned: • Recombination in mammalian cells is very sensitive to small degrees of sequence divergence -- a single mismatch matters • Not all mismatches inhibit recombination to the same degree (C-C or G-G mispairs strongly inhibit recombination) • The minimal amount of contiguous homology needed for efficient recombination is ~150 bp

9. Once recombination initiates (within homology) can it stray into adjacent mismatched sequences? Will any “blue” homeologous DNA end up in conversion tracts?

10. Can this happen? X X Also -- will the presence of the blue DNA suppress recombination?

11. Mismatches (blue homeologous DNA) did not reduce recombination rate But… Strikingly, 80 out of 81 recombinants from pHYB12-8 or pHYB21A contained no “blue” homeologous sequence in conversion tracts – not one single mismatch was transferred!

12. X X But what about this? X X This almost never happens:

13. 11 out of 39 recombinants contained the entire 60 bp patch of homeology in the conversion tract!

14. How is homeologous recombination suppressed?

15. How is homeologous recombination suppressed?

16. Conclusions • Mismatched recombination intermediates are established and exist transiently. • Avoidance of homeologous recombination does not involve wholesale destruction of mismatched intermediates. Rather, mismatched intermediates apparently are rejected by “unwinding.” • Rejection of mismatched DNA intermediates is driven by a search for localized homology and not by a response against mismatches per se.

17. What are the players that ensure high fidelity of recombination? Mismatch Repair?

18. 1 2 3 4 5 Msh2 I-SceI I-SceI Knockdown of MSH2 in mouse cells does not promote homeologous recombination

19. 1 2 3 4 5 p53 I-SceI I-SceI Knockdown of p53 in mouse cells does not promote homeologous recombination

20. I-SceI I-SceI Substrates to measure homologous and homeologous recombination in “any cell” : Homologous donor (pLB4): tk1 hyg tk1 neo H H B B Homeologous donor (pBR3): tk2 hyg tk1 neo H H B B

21. Adapted from Kass and Jasin (2010) FEBS Letters 584:3703-3708

22. How about RecQ family DNA helicases? From Brosh and Bohr (2007) Nucleic Acids Res. 35: 7527-7544

23. 3’-5 Exonuclease 3’-5’ helicase activity ATPase Nuclear targeting RecQ helicases

25. Normal BLM BLM deficient, deficient complemented BLM Does BLM deficiency promote homeologous recombination?

26. BLM deficiency appears to promote homeologous recombination •Difference between BLM deficient and complemented lines is significant (p = 1.71 x 10-4) • Also, 0 out of 71 DSB repair events recovered from normal human fibroblasts were homeologous recombinants (p = 1.18 x 10-5)

27. Mode of resolution of recombination can impact genome stability Gene conversions are fine, but crossovers can lead to LOH, deletions, inversions, or possibly other rearrangements.

28. conversion tract Maybe not so good Homologous Recombination is an Accurate Means of Double-Strand Break Repair Good

29. Chen et al., Nature Reviews Genetics 8, 762-775 (October 2007)

30. Knockdown of BLM doesn’t effect colony recovery following DSB induction The frequency of G418R colonies following DSB induction was similar for cells transfected with no siRNA, transfected with a non-silencing control siRNA, or transfected with either BLM-1 or BLM-3 siRNA that targeted BLM expression.

31. gene conversion tract X H H donor tk hyg tk neo 2.5 kb 5.9 kb X H tk neo 3.9 kb Gene conversion: Crossover:

32. + BLM-3 siRNA + Nonsilencing siRNA 5.9 kb - 3.9 kb - crossover 2.5 kb - gene conversion HR events were first identified by screening PCR products by AluI digestion, and then gene conversions were distinguished from crossovers by a double digest with HindIII (H) and XbaI (X) and Southern blotting using a tk probe. Shown are some representative results.

33. Knockdown of BLM increases homology-directed repair, and specifically increases crossover events.

34. Holliday junction dissolution? Promotion of SDSA? Unanswered: What is the mechanism by which BLM normally suppresses crossovers?

35. Werner cell line 1 Werner cell line 2 But 53 other clones (out of 105) were weird.

36. Also studying DSB repair in Werner cells

37. Translocation associated with NHEJ

38. Future studies: • Continue to investigate the roles of RecQ helicases using RNA interference and cells with RecQ null mutations. • Re-examine the roles of MMR, p53 and RecQ helicases in “telomerized” human cells • Investigate the role of nuclear paraspeckles in the maintenance of genome stability. (Collaboration with Doug Pittman.)

39. Acknowledgments Current lab members: Yibin Wang Shen Li Barbara C. Waldman Former members: Vik Bhattacharjee Laura Bannister Yunfu Lin Edie Goldsmith Vimala Kaza Krissy Smith Funding provided by:

41. DSB induction neo t k Repair (error-prone) t Functional fusion gene (G418R) neo k neo I-SceI Nonfunctional fusion gene (G418S ) neo tk